Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

Erythroid differentiation and the Na+,K+-pump in ouabain-sensitive and ouabain-resistant Friend erythroleukemia cell lines

The selection and biochemical characterization of ouabain-resistant erythroleukemia cell lines are described. Treatment of ouabain-resistant Friend erythroleukemia cell (FLC) lines with 1 mM ouabain demonstrated a reduced ouabain-sensitive 86Rb+-uptake after Na+-preloading in comparison with ouabain-sensitive cells. The ouabain- and diuretic (piretanide)-insensitive component of the 86Rb+-uptake (residual influx) was significantly enhanced in the ouabain-resistant FLC clones. Measurements of the Na+,K+-ATPase activity (E.C. 3.6.1.3) in plasma membrane preparations of the ouabain-resistant FLC clone B6/2 indicated that a ouabain-resistant Na+,K+-ATPase activity of about 20% of the total enzyme activity existed in the presence of 1 mM ouabain. Further experiments showed that the Na+,K+-ion-gradient in ouabain-resistant B6/2 cells was unaffected by ouabain exposure whereas the gradient collapsed in wild type 12 N cells. Another property of the ouabain-resistant cell lines was a decrease of the 86Rb+-uptake due to the Na+,K+, 2Cl(-)-cotransport system measured as piretanide-sensitive 86Rb+-uptake. The data on ion transport mechanisms in QuaR and QuaS FLC are discussed with respect to mutagen-induced and spontaneous cellular ouabain resistance. In addition, the role of altered ion transport mechanisms is considered for induced erythroid differentiation.     

Differentiation between serum stimulation of ouabain-resistant and sensitive Rb influx in quiescent NIH 3T3 cells

Summary The addition of serum to quiescent NIH 3T3 mouse cell cultures resulted in a 10- to 20-fold increase of Rb influx which was resistant to ouabain, and only a three- to fourfold activation of ouabain-sensitive Rb influx. Stimulation of the ouabain-resistant Rb influx following serum addition reached its maximum within 2 min. The stimulation of ouabain-resistant Rb influx was a result ofV _m increase while theK _m for Rb was unchanged. Ouabain-resistant Rb influx, after serum addition, was resistant to amiloride and sensitive to ethacrinic acid. Replacing chloride in the medium by NO₃ ^–, CO₃ ^– and CH₃COO^– resulted in a drastic decrease in the ouabain-resistant Rb influx. It appeared, therefore, that the ouabain-resistant Rb influx in NIH 3T3 cells was Cl^–-dependent.     

Phenotypic characterization of ouabain-resistant Aedes albopictus cells.

The phenotype of a ouabain-resistant Aedes albopictus cell line has been partially characterized. Treatment of ouabain-sensitive cells with 0.005-1.0 mM ouabain resulted in an 80% reduction in the uptake of 86rubidium (86Rb+), an ion with an affinity for the K+ pump binding site; ouabain-resistant cells showed only a 40% reduction with 1.0 mM ouabain. When ouabain-sensitive cells were incubated in the presence of ouabain (0.1 mM) for one and one-half to three hours, the molar ratio of intracellular Na+/K+ rose from 0.2 to 4.2. In ouabain-resistant cells, a similar treatment had very little effect. Based on [3H] ouabain-binding studies, ouabain-resistant cells were estimated to have 60% fewer binding sites per cell than ouabain-sensitive cells. The spontaneous mutation rate from ouabain sensitivity to ouabain resistance was calculated to be 1-6 x 10(-8) mutations/cell/generation, a value similar to that reported for mammalian cells at the analogous locus.     

Fibroblast growth factor induces a transient net K+ influx carried by the bumetanide-sensitive transporter in quiescent BALB/c 3T3 fibroblasts

The bumetanide-sensitive transport system performed a net efflux of K+ in serum deprived quiescent cells. The addition of partially purified fibroblast growth factor (FGF) to G0/G1 phase 3T3 fibroblasts induced a transient net influx of K+, carried out by the bumetanide-sensitive transport system for 2-6 minutes. The stimulation of the bumetanide-sensitive K+ influx by FGF was followed by stimulation of the ouabain-sensitive K+ influx. In addition, both the bumetanide-sensitive and the ouabain-sensitive K+ influxes were found to be similarly stimulated when the G0/G1 3T3 cells were treated with insulin. These results suggest that growth factors such as FGF and insulin induce a change in the action of the bumetanide-sensitive transporter from performing net K+ efflux along its concentration gradient to an uphill transport pumping of K+ into the cell. We propose, therefore, that the bumetanide-sensitive transporter contributes to the increase in the intracellular K+ (and probable Na+) stimulated by growth factors such as FGF and insulin in early G1 phase of the cell cycle.     

Transfer of potassium. A new measure of cell-cell coupling

Mammalian cells of different species differ in sensitivity to ouabain. This sensitivity is expressed as reduced intracellular K+ content, reduced rates of protein synthesis, and cessation of cell multiplication. Using 86Rb+ as a measure of intracellular K+, we found higher levels of radioactivity in mixtures of ouabain-sensitive and -resistant cells cultured in the presence of ouabain than predicted from pure cultures of the two component cell types. The simplest explanation is that K+ and 86Rb+ are being transferred from ouabain-resistant to ouabain-sensitive cells, enhancing the total intracellular 86Rb+ in the culture. A function, "index of cooperation," expresses this enhancement as a number ranging from 0 to 1, and permits comparisons to be made under various culture conditions and using various cell types. An index of cooperation greater than 0 requires cell contact, since no enhancement occurs when contact between two cell types in the same culture is prevented. The index of cooperation for a number of different cell combinations agrees with other measures of cell-cell interaction associated with gap junctions, such as electrical coupling and metabolic cooperation. Coculture of ouabain-sensitive and ouabain-resistant cells in the presence of ouabain also leads to restoration of the capacity for protein synthesis. Autoradiography shows that this restoration occurs in the sensitive cell type and is dependent upon contact with ouabain-resistant cells. Furthermore, sensitive cells are able to multiply in the presence of ouabain when cocultured with resistant cells. Thus K+, presumably transferred to sensitive cells through gap junctions, is able to counteract the toxic effects of ouabain on intracellular K+ levels and protein synthesis, and to restore growth.     

Cyclosporin A inhibits long-term activation of Na+,K+ pump in phytohemagglutinin-stimulated human lymphocytes

The effect of the immunosuppressive drug cyclosporin A (CsA) on the K (Rb) influx, intracellular K and Na contents, and on the major parameters of lymphocyte activation have been investigated in human peripheral blood lymphocytes activated by phytohemagglutinin (PHA). CsA suppressed protein, RNA, DNA syntheses and cell proliferation by 49.8 +/- 4.3, 67.6 +/- 10.1, 60.4 +/- 5.3 and 60.0 +/- 5.1%, respectively (n = 10) within 48 h. It also inhibited the late long-term Na+,K+ pump activation, as determined from the ouabain-sensitive Rb uptake, and prevented the increase in the intracellular K content at the late stages of G0/G1/S progression. Cyclosporin A did not affect the early transient pump activation, the dynamics, of ouabain-resistant influxes and the intracellular Na content in PHA-activated lymphocytes. When added 1 h after PHA, CsA neither affected the activation of the pump-mediated Rb influxes nor the increase in the intracellular K content. It is concluded that in activated human lymphocytes, the long-term activation of Na+,K+ pump associated with the mitogen-induced blast transformation, as well as the late increase in K content depend on the T-cell growth factor interleukin-2.     

Intracellular ionic changes in normal and transformed human fibroblasts after extracellular Ca2+ deprivation.

The lowering of extracellular Ca2+ concentration in the growth medium reversibly blocks normal, but not SV40-transformed WI38 diploid fibroblasts in the early G1/G0 phase of the cell cycle. This growth response is characterized by specific changes in ionic content and transport. Ca2+ deprivation (0.03 mM) has little effect on the K+ content of either normal or transformed cells. Na+ content, however, is increased nearly 2-fold in the normal cells. This increase is presumably due to a 3-fold increase in unidirectional Na+ influx in Ca2+-deprived cells. The increased intracellular Na+ also gives rise to a nearly 3-fold enhancement of the active (ouabain-sensitive) Na+ efflux. Ca2+ deprivation causes only slight increases in Na+ influx, ouabain-sensitive Na+ efflux and intracellular Na+ in the transformed cell. In contrast, the transformed cells lose nearly 60% of their intracellular Ca2+ on deprivation, whereas normal WI38 cells lose only 10%. The data suggest that the growth arrest exhibited by the normal cell but not the transformed cell may be related to different membrane-transport and permeability changes in response to Ca2+ deprivation.     

Stimulation of K+ transport systems by Ha-ras

The expression of Ha-ras in quiescent NIH3T3 cells carrying a glucocorticoid-inducible human Ha-ras gene (Val-Gly mutation at codon 12) stimulates total 86Rb+ influx. This effect is predominantly due to an elevated 86Rb+ uptake through an ouabain-resistant, furosemide-sensitive system. The ouabain-sensitive Na+/K(+)-ATPase is less affected. The transport which is resistant to both inhibitors is not altered by Ha-ras. Overexpression of the Ha-ras proto-oncogene causes only a marginal increase in total 86Rb+ uptake. The stimulation of the furosemide-sensitive influx by Ha-ras is paralleled by an increase in mean cell volume which can be inhibited by furosemide. A rapid stimulation of the furosemide-sensitive Rb+ influx is also observed after addition of bombesin to growth-arrested cells. Furosemide inhibits the mitogenic response after expression of Ha-ras or addition of bombesin. Both the Ha-ras and the bombesin-induced stimulation of the furosemide-sensitive Rb+ transport can be blocked by protein kinase C depletion or the protein kinase C inhibitor staurosporine. In contrast to bombesin-induced phosphatidylinositol-4,5-bisphosphate hydrolysis which is down-modulated by Ha-ras, the stimulation of the furosemide-sensitive Rb+ influx by bombesin is elevated in Ha-ras-expressing cells. This is in accordance with the increased mitogenic activity of bombesin in Ha-ras-expressing cells.     

Comparative study of the functional expression of the Na/K-pump in human lymphocytes, activated by phytohemagglutinin, phorbol ester, ionomycin, and interleukin-2

Rubidium (Rb) influxes via Na/K pump and ouabain-resistant pathways, and protein, RNA and DNA syntheses have been studied in human blood lymphocytes during the cell transit from quiescence to proliferation. In lymphocytes, stimulated either by phytohemagglutinin (PHA), phorbol 12,13-dibutyrate (PDBu) calcium ionophore, ionomycin, or by PDBu and interleukin-2 (IL-2), the late stages of the G0/G1-->S transit are accompanied by sustained 3-fold increased ouabain-sensitive Rb influxes. Using a two-pulse activation by a brief (1 h) PDBu/ionomycin treatment, followed by incubation with PDBu or IL-2 for 48 h, it has been found that both IL-2 and IL-2 receptor (IL-2R) are necessary for a long-term enhancement of Na/K pump. When present at the early stage of PDBu and ionomycin induction, cyclosporin A (CsA, 1 microgram/ml) inhibits the late increase in pump-mediated Rb influxes. However, when applied after the competence induction, CsA produced no effect on the flux increase at the progression stage. It is concluded that in activated human lymphocytes the functional expression of Na/K pump may by associated with IL-2-dependent progression of competent cells in the cell cycle.     

Glucose reduces both Rb+ influx and efflux in pancreatic islet cells

Microdissected, beta-cell-rich pancreatic islets from ob/ob mice were used in studies of 86Rb+ transport. D-Glucose (20 mM) induced a biphasic reduction in 86Rb+ efflux. The reduction stabilized within 10 min at 34% of the efflux rate at zero glucose. The initial 86Rb+ uptake (5 min) was dose-dependently reduced by ouabain with maximum inhibition at 1 mM. D-Glucose (20 mM) did not affect the ouabain-sensitive 86Rb+ influx but markedly reduced (48%) the ouabain-resistant isotope influx. The results suggest that D-glucose does not affect the Na+/K+ pump in pancreatic beta-cells and that the glucose-sensitive K+-transporting modalities (K+ channels) in the beta-cells can mediate both inward and outward K+ flux.     

Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells

Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle.     

Radioactive 86rubidium influx into red blood cells in essential hypertension in relation to the plasma renin activity

Changes in several mechanisms of sodium transport across the cell membranes are described in essential hypertension. We studied ouabain-sensitive and insensitive 86Rb+ influx into the red blood cells (RBC) of 16 healthy controls and 51 patients with essential hypertension (EH) divided according to their plasma renin activity (PRA) in 3 groups: 11 patients with high PRA (HREH), 18 patients with normal PRA (NREH) and 22 patients with low PRA (LREH). In addition to studying 86RB+ uptake by patients RBC, we tested also the effect of the patients' sera on 86Rb+ influx into the RBC of healthy subjects. Red blood cells of patients with HREH and NREH had lower ouabain-sensitive 86Rb+ influx in comparison with controls. No significant differences were found between these hypertensive groups. In contrast 86Rb+ uptake by the RBC of LREH patients was always higher than in controls or HREH and NREH. It was chiefly the ouabain-sensitive component that was raised, but some increase in ouabain-insensitive 86Rb+ influx also could be seen. The serum of patients with HREH and NREH, when incubated with RBC of healthy controls, lowered their ouabain-sensitive 86Rb+ influx. The decrease was more pronounced in NREH than in HREH group. Plasma from LREH patients increased both ouabain-sensitive and ouabain-insensitive 86Rb+ influx into the control RBC. These findings indicate that there may be differences in the sodium/potassium transport mechanisms across the cell membrane in various kinds of EH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of a Na+/K+/Cl- cotransporter in alkylating agent-sensitive L1210 murine leukemia cells

The mode of influx of 86Rb+, a K+ congener, to exponentially proliferating L1210 murine leukemia cells, incubated in a Krebs-Ringer buffer, has been characterised. The influx was composed of a ouabain-sensitive fraction (approx. 40%), a loop diuretic-sensitive fraction (approx. 40%) and a fraction which was insensitive to both types of inhibitor (approx. 15%). The fraction of ouabain-insensitive 86Rb+ influx, which was fully inhibited by furosemide (1 mM) or bumetanide (100 microM), was completely inhibited when Cl- was completely substituted by nitrate or gluconate ions, but was slightly (29 +/- 12%) stimulated if the Cl- was substituted by Br-. The substitution of Na+ by Li+, choline or tetramethylammonium ions inhibited the loop diuretic-sensitive fraction of 86Rb+ uptake. These results suggested that a component of 86Rb+ influx to L1210 cells was mediated via a Na+/K+/Cl- cotransporter. 86Rb+ efflux from L1210 cells which had been equilibrated with 86Rb+ and incubated in the presence or absence of 1 mM ouabain, was insensitive to the loop diuretics. Additionally, efflux rates were found to be independent of the external concentration of K+, suggesting that efflux was not mediated by K+-K+ exchange. The initial rate of 86Rb+ influx to L1210 cells in the plateau phase of growth was reduced to 44% of that of exponentially dividing cells, the reduction being accounted for by significant decreases in both ouabain- and loop diuretic-sensitive influx; these cells were reduced in volume compared to cells in the exponential phase of cell growth. In cells which had been deprived of serum for 18 h, and which showed an increase of the proportion of cells in the G1 phase of the cell cycle, the addition of serum stimulated an immediate increase in the furosemide-sensitive component of 86Rb+ influx. Diuretic-sensitive 86Rb+ influx was not altered by the incubation of the cells with 100 microM dibutyryl cyclic AMP, but was inhibited by 10 microM of the cross-linking agent nitrogen mustard (bis(2-chloro-ethyl)methylamine, HN2).     

CV-1 cell recipients of the mouse ouabain resistance gene express a ouabain-insensitive Na,K-ATPase after growth in cardioactive steroids

The cation-transporting activity and Na,K-ATPase activity of CV-1 cell recipients of the mouse ouabain resistance gene (ouaR6, or OR6 cells; see Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1489-1493) have been further characterized. OR6 cells grown in strophanthidin (a cardiac aglycon which may be removed rapidly from the Na,K-ATPase) possess both ouabain-sensitive and -insensitive 86Rb+ uptake activities. The ouabain-sensitive 86Rb+ uptake activity of these cells (OR6-S cells) exhibits the same Ki for ouabain as that of the CV-1 parent cells (Ki(app) = 3 x 10(-7) M ouabain), but accounts for only approximately 30% of total 86Rb+ uptake into Na+-loaded OR6-S cells, compared to 80% for CV-1 cells. Most of the ouabain-resistant 86Rb+ uptake in OR6-S cells is dependent on internal Na+ and is insensitive to furosemide, suggesting that it is due to an ouabain-resistant Na,K pump. In OR6-S cell lysates, 50% of Na+-dependent ATPase activity is insensitive to 1 mM ouabain, compared to less than 5% in CV-1 cell lysates. In addition, purified plasma membranes from OR6-S cells contain a 100-kDa protein which is transiently phosphorylated by ATP in an Na+-dependent, K+-sensitive manner, like the alpha subunit of the CV-1 Na,K-ATPase and the canine renal Na,K-ATPase, but which is unaffected by preincubation in 1 mM ouabain. All of these data suggest that OR6-S cells possess a ouabain-insensitive Na,K pump with characteristics similar to the ouabain-sensitive pump of CV-1 parent cells. Since the mouse ouabain resistance gene does not encode either subunit of the Na,K-ATPase, these results suggest that the ouabain resistance gene product may modify the ouabain sensitivity of the endogenous CV-1 Na,K pump.     

Barium mimics the effect of D-glucose on 86Rb+ fluxes in mouse pancreatic beta-cells

The interaction between Ba2+, furosemide and D-glucose on 86Rb+ fluxes in ob/ob mouse islets was investigated. Ba2+ (2 mM) significantly reduced the ouabain-resistant 86Rb+ influx, without affecting the ouabain-sensitive influx. D-Glucose (20 mM) reduced the 86Rb+ influx in the absence of Ba2+ (2 mM) but not in the presence of the cation. Furosemide, an inhibitor of Na+, K+, Cl- co-transport, reduced the 86Rb+ influx and the effect was partly additive to the effect of 2 mM Ba2+. When the islets were preincubated with Ba2+ (2 mM) the specific effect of 1 mM furosemide on the 86Rb+ influx was reduced, whereas, in acute experiments, Ba2+ (2 mM) did not affect the specific effect of furosemide on 86Rb+ influx. 86Rb+ efflux from preloaded islets was significantly reduced by 2 mM Ba2+ and during the first 5 min of ion efflux the effect of the combination of 2 mM Ba2+ and 1 mM furosemide was stronger than the effect of Ba2+ alone. The data show that Ba2+ reduces 86Rb+ fluxes in the beta-cells and suggest that this is mainly mediated by inhibition of K+ channels in the beta-cell plasma membrane. Long-term exposure to Ba2+ may also reduce the activity of the Na+, K+, Cl- co-transport system. The effect of Ba2+ on K+ channels may help to explain the stimulatory effect on insulin release in the absence of nutrient secretagogues.     

Functional expression of the Na/K pump is controlled via a cyclosporin A-sensitive signalling pathway in activated human lymphocytes.

An immunosuppressant cyclosporin A (CsA) inhibits T-cell proliferation by blocking the nuclear factor of activated T-cells (NFAT) required for expression of the interleukin-2 (IL-2) gene. This work has demonstrated for the first time that in human blood lymphocytes (HBLs) activated by phytohemagglutinin (PHA), CsA at anti-proliferative doses inhibits the late sustained increase in ouabain-sensitive Rb(K) influxes, which accompanies the growth phase of G0/G1/S transition. CsA affects neither the initial, transient activation of the pump in response to PHA nor the ouabain-resistant ion fluxes during cell cycle progression. When the HBLs were rendered competent to proliferate by phorbol 12,13-dibutyrate ester and ionomycin in the presence of CsA, the exogenous IL-2 did not bypass the initial inhibitory effect of CsA on the long-term pump enhancement. When applied after the competence induction, CsA produced no effect on the sustained increase in ouabain-sensitive Rb influxes during the IL-2-induced progression phase. These results indicate that in activated HBLs, (1) IL-2 is involved in functional expression of the Na/K pump during cell transition from quiescence to proliferation, (2) the cell cycle-associated upregulation of the pump is related to a CsA-sensitive signalling pathway.     

The effect of hyperthermia on the sodium-potassium pump in Chinese hamster ovary cells

The effect of hyperthermia on the Na+-K+ pump was determined by measuring influx and efflux of 86Rb+ in Chinese hamster ovary cells from 31 to 50 degrees C. The maximum initial rate of ouabain-sensitive influx increased with temperature between 31 and 45 degrees C although Km increased significantly above 37 degrees C, implying a diminished affinity of the transport protein for its substrate. The changes in the kinetics of influx at temperatures up to 45 degrees C were rapidly reversible on return to 37 degrees C. Above 45 degrees C an irreversible decrease in 86Rb+ uptake was observed. Efflux of 86Rb+ increased from 31 to 40 degrees C but above 43 degrees C showed a small but significant decrease. The study of 86Rb+ influx after varying times of exposure to elevated temperatures showed that the Na+-K+ pump remains functional in cells which are reproductively dead. We have shown that although the kinetics of K+ transport are sensitive to temperature changes in the range used in clinical hyperthermia, the inactivation of the Na+-K+ pump is not a primary event in cell killing.     

The functional unit of Na,K-ATPase is a monomeric alphabeta protomer

The ouabain-resistant and ouabain-sensitive alpha-subunit cRNAs in various molar ratios were injected into Xenopus oocytes together with cRNA for the beta-subunit. The ouabain-resistant ATPase activity, as well as ouabain-resistant Rb+ uptake, of the injected oocytes increased linearly with increasing the amount of cRNA for the ouabain-resistant alpha-subunit. When a functionless mutant was used instead of the ouabain-sensitive alpha-subunit, similar results were obtained in ATPase activity and Rb+ uptake. These results indicate that a monomeric alphabeta protomer is a functional unit of membrane-bound Na,K-ATPase, even if the enzyme exists structurally as a diprotomer or higher oligomers in membranes.     

The transport and distribution of monovalent cations during the blast transformation of human peripheral blood lymphocytes activated by phytohemagglutinin]

Potassium (rubidium) influx, sodium and potassium contents, as well as size distribution, DNA and protein contents and synthesis have been examined in PHA-activated human lymphocytes within 0.5-72 h. A complex set of ionic events was found to include at least two stages of the increase in potassium and sodium contents per g cell protein and in ouabain-sensitive potassium influx which are preceded by a decrease in potassium content by almost 17% within the first 2-5 h. The kinetics of potassium and sodium changes has own pattern for each of cations, thus indicating definite changes in the ouabain-resistant transport of potassium and sodium during the G0----G1----S progression. The late increase in potassium content per g cell protein was found to correlate with the growth in cell size. This finding confirms the rule which was stated earlier for other animal cells, i. e. cells that prepare to proliferate are to raise their potassium per g cell protein up to the level of 0.8-1.0 mmole (Vereninov. Marakhova, 1986).